Enhanced fluorescence resonance energy transfer between spectral variants of green fluorescent protein through zinc-site engineering

Although spectral variants of GFP should in theory be suited for fluorescen ce resonance energy transfer (FRET) and therefore suited for studies of pro tein-protein interactions, the unfavorable location of the fluorophore 15 A ngstrom deep inside the GFP molecule has especially impaired this applicati on. Here, metal-ion site engineering around the dimerization interface know n from the X-ray structure of OFF is applied to the cyan and the yellow spe ctral variant of GFP to stabilize the heterodimeric form of these molecules and thereby increase FRET signaling. The FRET signal, determined as the ra tio between the maximal emission for the yellow variant, 530 nm, and the cy an variant, 475 nm, during excitation of the cyan variant at 433 nm was inc reased up to 8-10-fold in the presence of 10(-4) M ZnCl2 by engineering of two symmetric metal-ion sites being either bidentate or tridentate. A simil ar increase in FRET signaling was however obtained in a pair of molecules i n which a single bidentate metal-ion site was generated by introducing a zi nc-binding residue in each of the two spectral variants of GFP and therefor e creating an obligate heterodimeric pair. It is concluded that FRET signal ing between spectral variants of GFP can be increased by stabilizing dimer formation and especially by favoring heterodimer formation in this case per formed by metal-ion site engineering.